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Disconnecting the Golgi ribbon from the centrosome prevents directional cell migration and ciliogenesis.

Hurtado L, Caballero C, Gavilan MP, Cardenas J, Bornens M, Rios RM - J. Cell Biol. (2011)

Bottom Line: We could thus demonstrate that breaking the polarity axis by perturbing GA positioning has a more dramatic effect on directional cell migration than disrupting the Golgi ribbon.Both features, however, were required for ciliogenesis.We thus identified AKAP450 as a key determinant of pericentrosomal Golgi ribbon integrity, positioning, and function in mammalian cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Señalización Celular, Centro Andaluz de Biología Molecular y Medicina Regenerativa-Consejo Superior de Investigaciones Científicas, 41092-Seville, Spain.

ABSTRACT
Mammalian cells exhibit a frequent pericentrosomal Golgi ribbon organization. In this paper, we show that two AKAP450 N-terminal fragments, both containing the Golgi-binding GM130-interacting domain of AKAP450, dissociated endogenous AKAP450 from the Golgi and inhibited microtubule (MT) nucleation at the Golgi without interfering with centrosomal activity. These two fragments had, however, strikingly different effects on both Golgi apparatus (GA) integrity and positioning, whereas the short fragment induced GA circularization and ribbon fragmentation, the large construct that encompasses an additional p150glued/MT-binding domain induced separation of the Golgi ribbon from the centrosome. These distinct phenotypes arose by specific interference of each fragment with either Golgi-dependent or centrosome-dependent stages of Golgi assembly. We could thus demonstrate that breaking the polarity axis by perturbing GA positioning has a more dramatic effect on directional cell migration than disrupting the Golgi ribbon. Both features, however, were required for ciliogenesis. We thus identified AKAP450 as a key determinant of pericentrosomal Golgi ribbon integrity, positioning, and function in mammalian cells.

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Membrane trafficking in AK1- and AK1B-transfected cells. (A) RPE-1 cells were transfected with VSVG-GFP (control) or cotransfected with VSVG-GFP and either flag-AK1 or flag-AK1B as indicated. VSVG-GFP was detected at the PM by incubating with the ectodomain antibody 8G5 during the last 30 min of a 60- or 100-min period after the shift at a permissive temperature. Cells were then fixed and stained as indicated (top images). 8G5 labeling was also quantified by using MetaMorph (n = 20 for each condition). Error bars indicate standard deviations. (B) Identical experiment as in A, but at different times after the permissive temperature shift, cells were fixed and stained as indicated. Arrows indicate the CTR, and arrowheads show the GA. (C, left) Video frames showing translocation of venus-NPY–containing vesicles in control cells, AK1-, or AK1B-expressing cells. (middle) Venus-NPY images recorded over 2 min were overlaid to visualize vesicle tracks. (right) Randomly selected particles were tracked for 2 min, and movement tracks are represented in different colors over a phase-contrast image of the recorded cell. Dashed lines indicate the cell border. Bars: (A and B) 10 µm; (C) 2.5 µm.
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fig7: Membrane trafficking in AK1- and AK1B-transfected cells. (A) RPE-1 cells were transfected with VSVG-GFP (control) or cotransfected with VSVG-GFP and either flag-AK1 or flag-AK1B as indicated. VSVG-GFP was detected at the PM by incubating with the ectodomain antibody 8G5 during the last 30 min of a 60- or 100-min period after the shift at a permissive temperature. Cells were then fixed and stained as indicated (top images). 8G5 labeling was also quantified by using MetaMorph (n = 20 for each condition). Error bars indicate standard deviations. (B) Identical experiment as in A, but at different times after the permissive temperature shift, cells were fixed and stained as indicated. Arrows indicate the CTR, and arrowheads show the GA. (C, left) Video frames showing translocation of venus-NPY–containing vesicles in control cells, AK1-, or AK1B-expressing cells. (middle) Venus-NPY images recorded over 2 min were overlaid to visualize vesicle tracks. (right) Randomly selected particles were tracked for 2 min, and movement tracks are represented in different colors over a phase-contrast image of the recorded cell. Dashed lines indicate the cell border. Bars: (A and B) 10 µm; (C) 2.5 µm.

Mentions: To address the significance of GA integrity or positioning in secretion, we first monitored ER to plasma membrane (PM) transport of the temperature-sensitive mutant of VSVG-GFP in control, AK1-, or AK1B-transfected cells (Fig. 7, A and B). VSVG-GFP was detected at the PM by incubating with an ectodomain antibody during the last 30 min of a 60- or 100-min period after the shift at a permissive temperature (Fig. 7 A). VSVG-GFP accumulated at the cell surface under all conditions. Quantification revealed no significant differences between control and transfected cells (Fig. 7 A, bottom). In parallel, transfected cells were fixed at different time points after the temperature shift (Fig. 7 B). Trafficking from the ER to the GA was apparently unaffected in mutant-expressing cells even when the GA was localized away from the CTR. It should be noted that endoplasmic reticulum exit sites (ERES) concentrated around the GA regardless of their positioning inside the cell (Fig. S5). VSVG-GFP was transported from the ER to the PM in both AK1- and AK1B-expressing cells, although a slight delay was apparent in VSVG transport from the GA to the PM (Fig. 7 B, right column). In agreement with previously published data (Miller et al., 2009; Yadav et al., 2009), our results indicate that neither Golgi ribbon integrity nor positioning are critical for global secretion.


Disconnecting the Golgi ribbon from the centrosome prevents directional cell migration and ciliogenesis.

Hurtado L, Caballero C, Gavilan MP, Cardenas J, Bornens M, Rios RM - J. Cell Biol. (2011)

Membrane trafficking in AK1- and AK1B-transfected cells. (A) RPE-1 cells were transfected with VSVG-GFP (control) or cotransfected with VSVG-GFP and either flag-AK1 or flag-AK1B as indicated. VSVG-GFP was detected at the PM by incubating with the ectodomain antibody 8G5 during the last 30 min of a 60- or 100-min period after the shift at a permissive temperature. Cells were then fixed and stained as indicated (top images). 8G5 labeling was also quantified by using MetaMorph (n = 20 for each condition). Error bars indicate standard deviations. (B) Identical experiment as in A, but at different times after the permissive temperature shift, cells were fixed and stained as indicated. Arrows indicate the CTR, and arrowheads show the GA. (C, left) Video frames showing translocation of venus-NPY–containing vesicles in control cells, AK1-, or AK1B-expressing cells. (middle) Venus-NPY images recorded over 2 min were overlaid to visualize vesicle tracks. (right) Randomly selected particles were tracked for 2 min, and movement tracks are represented in different colors over a phase-contrast image of the recorded cell. Dashed lines indicate the cell border. Bars: (A and B) 10 µm; (C) 2.5 µm.
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Related In: Results  -  Collection

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fig7: Membrane trafficking in AK1- and AK1B-transfected cells. (A) RPE-1 cells were transfected with VSVG-GFP (control) or cotransfected with VSVG-GFP and either flag-AK1 or flag-AK1B as indicated. VSVG-GFP was detected at the PM by incubating with the ectodomain antibody 8G5 during the last 30 min of a 60- or 100-min period after the shift at a permissive temperature. Cells were then fixed and stained as indicated (top images). 8G5 labeling was also quantified by using MetaMorph (n = 20 for each condition). Error bars indicate standard deviations. (B) Identical experiment as in A, but at different times after the permissive temperature shift, cells were fixed and stained as indicated. Arrows indicate the CTR, and arrowheads show the GA. (C, left) Video frames showing translocation of venus-NPY–containing vesicles in control cells, AK1-, or AK1B-expressing cells. (middle) Venus-NPY images recorded over 2 min were overlaid to visualize vesicle tracks. (right) Randomly selected particles were tracked for 2 min, and movement tracks are represented in different colors over a phase-contrast image of the recorded cell. Dashed lines indicate the cell border. Bars: (A and B) 10 µm; (C) 2.5 µm.
Mentions: To address the significance of GA integrity or positioning in secretion, we first monitored ER to plasma membrane (PM) transport of the temperature-sensitive mutant of VSVG-GFP in control, AK1-, or AK1B-transfected cells (Fig. 7, A and B). VSVG-GFP was detected at the PM by incubating with an ectodomain antibody during the last 30 min of a 60- or 100-min period after the shift at a permissive temperature (Fig. 7 A). VSVG-GFP accumulated at the cell surface under all conditions. Quantification revealed no significant differences between control and transfected cells (Fig. 7 A, bottom). In parallel, transfected cells were fixed at different time points after the temperature shift (Fig. 7 B). Trafficking from the ER to the GA was apparently unaffected in mutant-expressing cells even when the GA was localized away from the CTR. It should be noted that endoplasmic reticulum exit sites (ERES) concentrated around the GA regardless of their positioning inside the cell (Fig. S5). VSVG-GFP was transported from the ER to the PM in both AK1- and AK1B-expressing cells, although a slight delay was apparent in VSVG transport from the GA to the PM (Fig. 7 B, right column). In agreement with previously published data (Miller et al., 2009; Yadav et al., 2009), our results indicate that neither Golgi ribbon integrity nor positioning are critical for global secretion.

Bottom Line: We could thus demonstrate that breaking the polarity axis by perturbing GA positioning has a more dramatic effect on directional cell migration than disrupting the Golgi ribbon.Both features, however, were required for ciliogenesis.We thus identified AKAP450 as a key determinant of pericentrosomal Golgi ribbon integrity, positioning, and function in mammalian cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Señalización Celular, Centro Andaluz de Biología Molecular y Medicina Regenerativa-Consejo Superior de Investigaciones Científicas, 41092-Seville, Spain.

ABSTRACT
Mammalian cells exhibit a frequent pericentrosomal Golgi ribbon organization. In this paper, we show that two AKAP450 N-terminal fragments, both containing the Golgi-binding GM130-interacting domain of AKAP450, dissociated endogenous AKAP450 from the Golgi and inhibited microtubule (MT) nucleation at the Golgi without interfering with centrosomal activity. These two fragments had, however, strikingly different effects on both Golgi apparatus (GA) integrity and positioning, whereas the short fragment induced GA circularization and ribbon fragmentation, the large construct that encompasses an additional p150glued/MT-binding domain induced separation of the Golgi ribbon from the centrosome. These distinct phenotypes arose by specific interference of each fragment with either Golgi-dependent or centrosome-dependent stages of Golgi assembly. We could thus demonstrate that breaking the polarity axis by perturbing GA positioning has a more dramatic effect on directional cell migration than disrupting the Golgi ribbon. Both features, however, were required for ciliogenesis. We thus identified AKAP450 as a key determinant of pericentrosomal Golgi ribbon integrity, positioning, and function in mammalian cells.

Show MeSH
Related in: MedlinePlus